Methods for forming normal regenerated tissues, the normal regenerated tissues and methods for assessing sensitivities and so on

ABSTRACT

A normal regenerated tissue is formed by exposing to radiation a connective tissue or a supporting tissue originating in an organ to thereby form a feeder layer and then transplanting epithelial cells thereon to form a stratified structure. By conveniently and surely providing regenerated tissue by the 3-dimensional culture with the use of a human-origin normal tissue as a base, it is possible to construct systems for assessing effects and side effects of chemicals such as drugs or assessing sensitivities thereof with the use of regenerated tissues as models of corresponding tissues respectively.

TECHNICAL FIELD

The present invention relates to a method for forming a normalregenerated tissue, a normal regenerated tissue and a method forassessing sensitivity and the like.

BACKGROUND ART

While organ transplantation even after a cerebral death has not readilyis put into action in Japan, a 3-dimensional culture mainly of tissuestem (TS) cell is conducted constantly for the purpose of a regenerationmedical care. A use even of embryonic stem (ES) cell will be initiatedsoon although its ethical aspect is limited actually. Nevertheless, anyof these technologies is far from the achievement of regenerated organcapable of exerting functions sufficiently. On the other hand, a humanorgan and tissue bank is attempted to be established and maintained asan opponent to a genome-derived pharmaceutical developed bymanufacturers of medicines. Under such a circumstance, a formation ofregenerated tissue by stratifying human-derived normal tissues andinducing a differentiation in a more simple and reliable manner whencompared with a prior art is expected greatly and its realization is asignificant objective, since a system for predicting the effects and theside effects of, or a system for assaying the sensitivity to a chemicalsubstance mainly for a pharmaceutical can be constructed by obtaining arelevant organ model from the respective regenerated tissue.

Among current systems for assaying the effects of or the sensitivitiesto pharmaceuticals, a histoculture drug response assay (HDRA) using acollagen gel is known as a relatively effective means.

However, the HDRA has a problematically lower clinical true positiverate which is less than about 60%. Since the HDRA has a true negativerate of 80% or more, it is employed in a medical care practice only forscreening for a non-effective anti-cancer agent in US. It is notregarded to be a recognized custom-made therapy which allows a mosteffective drug to be selected. Nevertheless, it is regarded as a highlyadvanced medical technology in Japan, although most of clinicalpractitioners are disappointed by the fact that it can select aneffective drug at a probability as low as about 50%.

It fails also in assaying any side effect on normal tissuessimultaneously.

A known method other than the HDRA is a CG-DST (collagen gel dropletembedded culture sensitivity test). However, this method involves cancercells growth failure (no growth even in control) as high as 25% or moreand gives an unsuccessful result at a rate as seriously high as 30% ormore including the contamination rate, although its true positive rateis improved markedly to a level as high as 70 to 83%. Thus, whenmultiplying these two rates, the sensitivity can eventually be assayedonly in 60% of the patients. Also from the data of the ovarian cancer,the assumption may vary depending on the tissue types. The potency ofany side effect on normal tissues cannot be assayed also by this method.

Accordingly, any of the cancer therapy sensitivity tests of the priorart cannot predict the side effects on normal tissues simultaneously,even if it can evaluate the sensitivity of cancer cells or tissue to atherapy. It is far from an ideal custom-made therapy enabling theselection of a drug having fewer side effects.

Also in view for example of the current state described above, anassumption of the effects of and the sensitivity to a pharmaceuticalshould be enabled as soon as possible by obtaining a high leveldetection and evaluation system using regenerated tissues.

Accordingly, an objective of the invention is to overcome thelimitations and the problems associated with the prior art describedabove, to obtain regenerated tissues simply and reliably by a3-dimensional culture from a human-derived normal tissue as a base, andto provide, while utilizing the formers, a method for constructing asystem for predicting the effects and the side effects of a chemicalsubstance such as a pharmaceutical using thus regenerated tissues as arespective organ model or a system for predicting the sensitivity andthe like.

DISCLOSURE OF INVENTION

For solving the problems described above, the invention provides amethod for forming normal regenerated tissues comprising irradiating anorgan-derived connective tissue or its constituent cells or supportingtissue to form a feeder layer followed by transplanting epithelial cellsto form a stratified structure as a first aspect, a method for formingnormal regenerated tissues wherein the connective tissue or itsconstituent cells or supporting tissue and the epithelial tissue areorthotopic with regard to the organ-derived as a second aspect, a methodfor forming normal regenerated tissues wherein the connective tissue orits constituent cells or supporting tissue are at least any of theorgan-derived fibroblasts, endothelial cells or its constituent tissueas a third aspect, and a method for forming normal regenerated tissueswherein vascular endothelial cells are transplanted on organ-derivedfibroblasts and then an irradiation is effected to form a feeder layer,and then the epithelial cells are transplanted to form a stratifiedstructure as a fourth aspect.

The invention also provides a method for forming a normal regeneratedtissue wherein after transplanting the epithelial cells at least oneextracellular matrix is added to form the epithelial cells in astratified structure as a fifth aspect, a method for forming a normalregenerated tissue wherein the extracellular matrix is a structuralcomponent or adhesion molecule of an extracellular substrate as a sixthaspect, a method for forming a normal regenerated tissue wherein theextracellular matrix is at least one of collagen, elastin, proteoglycan,fibronectin, laminin and tenascin as a seventh aspect, a method forforming a normal regenerated tissue wherein after transplanting acollagen or collagen with fibronectin as well as laminin are added toeffect a gelatin whereby forming the epithelial cells in a stratifiedstructure as the eighth aspect, a method for forming a normalregenerated tissue wherein the connective tissue or its constituentcells or supporting tissue is co-cultured with at least one ofheterogenous constituent cells deriving from an orthotopic organ or aculture supernatant thereof and thereafter irradiated to form a feederlayer as a ninth aspect, a method for forming a normal regeneratedtissue wherein heterogenous constituent cells deriving from anorthotopic organ, epithelial cells and a culture supernatant thereof isco-cultured to form a stratified structure as a tenth aspect, and amethod for forming a normal regenerated tissue wherein the irradiationis an X-ray or γ-ray irradiation as an eleventh aspect, and a method forforming a normal regenerated tissue wherein the stratified structure ofthe epithelial cells are formed by a culture at a carbon dioxide gasconcentration of 5 to 15%, air concentration of 85 to 95% at a culturetemperature of 20 to 40° C. as a twelfth aspect.

The invention also provides a normal regenerated tissue comprising astratified structure of epithelial cells on a feeder layer from anorgan-derived connective tissue or constituent cells or supportingtissue as a thirteenth aspect, a normal regenerated tissue wherein theepithelial cells derive from nerve, oral mucosa, skin, bronchus, mammarygland, liver or kidney as a fourteenth aspect, a normal regeneratedtissue wherein the connective tissue or its constituent cells orsupporting tissue and the epithelial tissue are orthotopic with regardto the deriving organ as a fifteenth aspect, a normal regenerated tissuewherein the connective tissue or its constituent cells or supportingtissue are at least any of the organ-derived fibroblasts, endothelialcells or its constituent tissue as a sixteenth aspect, a normalregenerated tissue comprising a feeder layer consisting of organ-derivedfibroblasts and vascular endothelial cells and epithelial cells in astratified structure placed on the feeder layer as a seventeenth aspect,and a normal regenerated tissue wherein the epithelial cells are in astratified structure consisting of four or more layers as a eighteenthaspect.

The invention also provides a normal regenerated tissue comprising anormal regenerated tissue placed on a basal plate as a nineteenthaspect, a normal regenerated tissue wherein a culture medium or culturefluid is allowed to flow in contact with a normal regenerated tissue asa twentieth aspect, a normal regenerated tissue wherein a plural ofnormal regenerated tissues are allowed to pass through the channel ofthe culture medium or culture fluid as a twenty first aspect and anormal regenerated tissue which constitutes regenerated organ model bodyas a twenty second aspect.

The invention also provides a method for assessing a sensitivity ofcancer cells comprising inhibiting the proliferation of regeneratedepithelial cells by irradiating any normal regenerated tissue describedabove followed by transplanting the cancer cells on the epithelial cellsin a stratified structure followed by adding a chemical substance orirradiation as a twenty third aspect, a method wherein the assessment iseffected at a low oxygen concentration as a twenty fourth aspect, amethod comprising supplementing at least one of collagen or otherextracellular matrixes after transplanting cancer cells or transplantingcancer cells after supplementing at least one of collagen or otherextracellular matrixes, followed by assessing the sensitivity as atwenty fifth aspect.

The invention also provides a method for assessing an angiogeneticability comprising inhibiting the proliferation of regeneratedepithelial cells by irradiating any normal regenerated tissue describedabove followed by transplanting the cancer cells on the epithelial cellsin a stratified structure followed by supplementing at least one ofcollagen or other extracellular matrixes on which then vascularendothelial cells are transplanted followed by assessing theangiogenetic ability of the cancer cells in response to the addition ofa chemical substance as a twenty sixth aspect.

The invention also provides a method for assessing an angiogeneticability comprising inhibiting the proliferation of regeneratedepithelial cells by irradiating a normal regenerated tissue followed bytransplanting the cancer cells on the epithelial cells in a stratifiedstructure followed by mounting at least one of collagen or otherextracellular matrixes followed by inverting the entire structurewhereby assessing a motility or invasion ability for the purpose ofevaluating the effect of a metastasis or invasion inhibitor as a twentyseventh aspect.

The invention also provides a method for assessing a sensitivity of anormal regenerated tissue comprising an exposure of the tissue to achemical substance or irradiation as a twenty eighth aspect.

The invention also provides a method for assessing a gene transductioncomprising predicting the efficiency of the gene transduction in anormal regenerated tissue as a twenty ninth aspect.

The invention also provides a method of any of those listed abovewherein the irradiation for inhibiting the proliferation of regeneratedepithelial cells are an X-ray or γ-ray irradiation as a thirtiethaspect.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an outline exemplifying an inventive method;

FIG. 2 is an outline exemplifying a method to be added to the process ofFIG. 2;

FIG. 3 is a microscopic photograph showing the condition of a co-cultureof normal pulmonary fibroblasts and vascular endothelial cells beforethe X-ray irradiation at 20Gy;

FIG. 4 is a microscopic photograph showing the condition 24 hours afterthe transplantation of bronchial epithelial cells 24 hours after theirradiation described above;

FIG. 5 is a microscopic photograph showing the condition after 1 week;

FIG. 6 is a microscopic photograph showing the condition of thestratified structure having 4 layers or more although observed onlypartly;

FIG. 7 is a microscopic photograph showing regenerated bronchialepithelial cells in a normal regenerated tissue by an inventive method;

FIG. 8 is a microscopic photograph showing regenerated bronchialepithelial cells 3 days after the X-ray irradiation at 10Gy;

FIG. 9 is a microscopic photograph showing regenerated bronchialepithelial cells 3 days after the X-ray irradiation at 20Gy;

FIG. 10 is a microscopic photograph showing the condition of aregenerated bronchial epithelial cell layer formed by an inventivemethod which had been supplemented with a collagen gel and thentransplanted with human pulmonary cancer-derived AOI cells after threedays;

FIG. 11 is a microscopic photograph showing the condition immediatelyafter the X-ray irradiation at 20GY 3 hours after the transplantationsimilar to that in FIG. 10;

FIG. 12 is a microscopic photograph showing the condition 3 days afterthe irradiation in the case shown in FIG. 11;

FIG. 13 is a microscopic photograph showing the condition after theX-ray irradiation at 10Gy in the case shown in FIG. 12;

FIG. 14 is a microscopic photograph of renal proximal tubular epithelialcells (RPTEC) in a stratified structure in Embodiment 2;

FIG. 15 is a microscopic photograph of mesangium cells (NHMC) in astratified structure;

FIG. 16 is a microscopic photograph of prostatic epithelial cells (PrEc)in a stratified structure;

FIG. 17 is a microscopic photograph of neural progenitor cells (NHMP) ina stratified structure;

FIG. 18 is a microscopic photograph of human hepatocytes (NHeps) in astratified structure;

FIG. 19 is a microscopic photograph of mammary gland epithelial cells(HMEC) in a stratified structure;

FIGS. 20, 21, 22 and 23 show the results of the cancer therapysensitivity test by an MTT assay of the above-mentioned NHMP, PrEC, NHMCand PRTEC, respectively;

FIGS. 24, 25, 26, 27, 28 and 29 show the results of the cancer therapysensitivity test by an MTT assay of an NHBE in a stratified structure inEmbodiment 3 using SuSa (derived from skin) as a feeder layer;

FIGS. 30 and 31 show the results of the cancer therapy sensitivity testby an MTT assay of NHBE in a stratified structure using NHLF (derivedfrom lung) as a feeder layer;

FIGS. 32, 33 and 34 show the results of the cancer therapy sensitivitytest by an MTT assay of PrEc in a stratified structure using SuSa(derived from skin) as a feeder layer;

FIG. 35 is a microscopic photograph of PrEc regenerated in a stratifiedstructure with adding only a collagen in Embodiment 4. On the otherhand, FIG. 36 is a microscopic photograph using only a fibronectinwithout using a collagen;

FIGS. 37( a) and 37(b) are microscopic photographs obtained when addinga 1:1 or 1:2 mixture of a fibronectin and laminin to a collagen whenregenerating NHDF-Ad: human skin keratinized cells in a stratifiedstructure on a feeder layer of human skin-derived fibroblasts;

FIGS. 38( a) and 38(b) are microscopic photographs obtained when addingonly a collagen or adding a 2:1 mixture of a fibronectin and laminin;

FIGS. 39( a) and 39(b) are microscopic photographs obtained when addinga collagen together with a fibronectin and laminin in humanmelanoma-derived SK-Me126 cells invasion test of NHDF-Ad regenerated asa stratified structure in Embodiment 6;

FIGS. 40( a), 40(b), 41(a), 41(b), 42(a) and 42(b) are microscopicphotographs exemplified as Cases 1 to 12 showing the effects of theaddition of the fibronectin and laminin to the collagen in the PrECculture as a stratified structure in Embodiment 7;

FIGS. 43 and 44 show the results of the sensitivity test using humanrenal cancer-derived ACHN-L4 cells for each of a human renal proximaltubular epithelial cells (RPTEC) and mesangium cells (NHMC) eachregenerated in a stratified structure;

FIG. 45 shows the effect of the addition of the culture supernatant ofskin keratinized cell-derived NHDF-Ad cells for the RPTEC in astratified structure; and

FIGS. 46( a)-46(c) are microscopic photographs exemplifying the changein the sensitivity discussed above.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is characterized as described above, and itsembodiments are described below.

First, for describing the background of the invention briefly, thesignificance of the orthotopic transplantation has been emphasized by J.Fiedler in MD Anderson Cancer Center in 1970's and later through thestudies especially of cancer metastasis and spread all over the world.Thus, in a lung, there is an environment generated as a result of ahomeostasis attributable to lung-specific growth factors or humouralfactors such as cytokines as well as an adhesion between cells.Accordingly, it has been suggested that “under such an environment,lung-derived cells such as fibroblasts can exert their natural traitmost evidently”. Recently, his team rather reported that a fibroblastderived from each organ underwent a mutation specific thereto thus beingdifferent from each other. While this report has not been acceptedwidely, another recent report suggested that a subculture resulted inthe induction of p16 in a pulmonary fibroblast but not in a dermalfibroblast.

While taking these findings into account, we provide a far moreefficient novel 3-dimensional culture method when compared withconventional 3-dimensional methods employing an endogenousvirus-carrying mouse-derived NIH3T3 cell as a feeder layer (FL) oremploying only a collagen gel which is a ubiquitous extracellularmatrix. Our method utilizes the nature of the orthotopic transplantationdescribed above while taking advantage of the fact that cancer cells cannot keep existing exclusively in a living body without any normaltissue, the fact that almost no noise is generated in a gene expressionprofile by irradiated cells or FL, the fact that normal (epithelial)cells, apart from cancer cells, hardly undergoes a proliferation in astratified structure usually on a gel, and the fact that irradiatedcells or FL causes a negligible noise on an MTT assay.

Thus, the present invention is characterized by irradiating anorgan-derived connective tissue or its constituent cells or supportingtissue to form a feeder layer followed by transplanting epithelial cellsto form a stratified structure, and in this case it is a matter ofcourse that the connective tissue or supporting tissue is a producer ofa growth factor or cytokine.

A representative which plays a role of a connective tissue or itsconstituent cells or supporting tissue may for example be organ-derivedfibroblasts, endothelial cells or its constituent tissue. Among thoselisted above, fibroblasts and vascular endothelia can be co-cultured inthe invention. Otherwise, a glia cell, smooth muscle cell or a musclesuch as a myocardium can be mentioned.

Especially in the invention, it is an important embodiment that aconnective tissue or its constituent cells or supporting tissues andepithelial cells are orthotopic with regard to the deriving organ, whichis thus an identical skin, kidney, bronchus, nerve and the like. Such anorthotopicity ensures an efficient formation of a stratified structureof the epithelial cells.

More typically, the invention provides a method for forming a normalregenerated tissue wherein vascular endothelial cells are transplantedon organ-derived fibroblasts in a medium and then an irradiation iseffected to form a feeder layer, and then the epithelial cells aretransplanted to form a stratified structure.

An inventive method is characterized also in that after transplantingthe epithelial cells at least one extracellular matrix is added to formthe epithelial cells in a stratified structure, and also in that theextracellular matrix is a structural component or adhesion molecule ofan extracellular substrate, more typically at least one of collagen,elastin, proteoglycan, fibronectin, laminin and tenascin. Moreover, thestrafing efficiency is improved further by adding growth factors derivedfrom each organ.

In a preferred embodiment, a collagen or collagen with fibronectin aswell as laminin are added after transplanting to form the epithelialcells in a stratified structure, or the irradiation is an X-ray or γ-rayirradiation. Furthermore, it is also preferable that the connectivetissue or its constituent cells or supporting tissue is co-cultured withat least one of a heterogenous constituent cells deriving from anorthotopic organ or a culture supernatant thereof and thereafterirradiated to form a feeder layer, that heterogenous constituent cellsderiving from an orthotopic organ, epithelial cells and a culturesupernatant thereof are co-cultured to form a stratified structure, andthat the stratified structure of the epithelial cells are formed by aculture at a carbon dioxide gas concentration of 5 to 15%, airconcentration of 85 to 95% at a culture temperature of 20 to 40° C.

According to an inventive method described above, a normal regeneratedtissue comprising a stratified structure of epithelial cells on a feederlayer from an organ-derived connective tissue or constituent cells orsupporting tissue is obtained.

In such a case, the epithelial cell layer may be of any type, such asthose deriving from nerve, oral mucosa, skin, bronchus, mammary gland,liver or kidney.

According to the invention, the stratified structure consisting ofthree, four, five or more layers, i.e., about four or more layers isreadily available. The resultant normal regenerated tissue may be placedon a basal plate, or may be one wherein a culture medium or culturefluid is allowed to flow in contact with a normal regenerated tissue orone which constitutes regenerated organ model body.

Also according to the invention, a method for predicting a sensitivityof cancer cells comprising inhibiting the proliferation of regeneratedepithelial cells by irradiating a normal regenerated tissue followed bytransplanting the cancer cells on the epithelial cells in a stratifiedstructure followed by adding a chemical substance or irradiation, andits typical example which is a method wherein the prediction is effectedat a low oxygen concentration as well as a method comprisingsupplementing at least one of collagen or other extracellular matrixesafter transplanting cancer cells or transplanting cancer cells aftersupplementing at least one of collagen or other extracellular matrixes,followed by assessing the sensitivity are provided.

Moreover, a method for predicting an angiogenetic ability comprisinginhibiting the proliferation of regenerated epithelial cells byirradiating a normal regenerated tissue followed by transplanting thecancer cells on the epithelial cells in a stratified structure followedby supplementing at least one of collagen or other extracellularmatrixes on which then vascular endothelial cells are transplantedfollowed by predicting the angiogenetic ability of the cancer cells inresponse to the addition of a chemical substance, a method forpredicting an angiogenetic ability comprising inhibiting theproliferation of regenerated epithelial cells by irradiating a normalregenerated tissue followed by transplanting the cancer cells on theepithelial cells in a stratified structure followed by mounting at leastone of collagen or other extracellular matrixes followed by invertingthe entire structure whereby assessing a migrating or invasion ability,a method for assessing a sensitivity of a normal regenerated tissuecomprising an exposure of the tissue to a chemical substance orirradiation, and a method for assessing a gene transduction comprisingassessing the efficiency of the gene transduction in a normalregenerated tissue are provided.

The embodiments of the invention are exemplified below with referring tothe attached figures which outline the invention.

Embodiment 1

For example with regard to a system for assessing sensitivity, amultiwell dish such as 6-well, 12-well or 24-well dish may be employed.First, fibroblasts and vascular endothelial cells derived from anintended organ is isolated from a surgically excised tissue by means ofa treatment with a collagenase and trypsin in a mild manner such thatthe bulk of cells are not separated into individual cells and thenidentified using a monoclonal antibody, cell sorter and the like. Then,for example as shown in FIG. 1, a 15% fetal calf serum-supplementedmaintenance medium (1) is used to inoculate about 0.5×105 cells/cm² ofthe fibroblasts (ratio:10) (2) with the vascular endothelial cells(ratio:1) (3), which is then irradiated within the period of about 6 to24 hours with a preconditioning dose (20Gy or less) of an X-ray (4) formaintaining the tissue structure as a fundamental layer over about 2weeks while suppressing the proliferation of the fibroblasts to form anFL (5).

On this FL (5), epithelial cells layer (about 1.0×10⁵ cells/cm²) (6)separated from the identical excised tissue by a mild disperse treatmentis transplanted. The epithelial cells (6) are going to be stratified3-dimensionally to become a regenerated epithelium (7). When the donoris an elderly human or a human having a heavily smoking history, the toplayer was covered with a sterilized and ice-cold liquid collagen (8)[prepared by diluting with a medium supplemented with relatively lowlevels of various growth factors (the growth factors at concentrationsas low as about ½ or less of the levels in the maintenance medium)],which was then gelled by warning the temperature for example to 37° C.When maintaining the regenerated tissue, the carbonate gas level iselevated slightly when compared with an ordinary level, such as 10%, andthe culture is conducted at an air level of 90% at 37° C. Alsococultured are a carbonate gas level of 5 to 15%, air level of 85 to 95%and a temperature of 20 to 40° C. During this culture, the culturemedium is replaced for example three times a week. At the time pointwithin about 2 weeks when the stratified structure acquired about 4layers or more i.e., 4 layers or 3 layers or more, the major effects,side effects or toxic effects on a normal tissue are evaluated after thetreatment with various combinations of chemicals at respectiveconcentrations or within about several hours to 24 hours after theirradiation using an MTT assay, gene expression profile or proteinprofile.

Table 1 shows the compositions of a medium assayed to be employed beforethe step for forming a stratified structure such as an NHBH (e.g., formammary gland epithelium or bronchial epithelium) and a high-factor NHBE(for a special growth promotion of bronchial epithelium and the like)and a medium assayed to be employed in forming a stratified structuresuch as the maintenance medium.

TABLE 1 Total volume of maintenance High-factor medium 420 ml NHBE NHBEF-12 100 ml/420 ml Transferrin 5.0 μg/ml 10 μg/ml 10 μg/ml Insulin 5.0μg/ml 5 μg/ml 5 μg/ml Hydrocrtizon 0.4 μg/ml 0.5 μg/ml 0.5 μg/mlTriiodothyronin 2 × 10⁻⁹ M 6.51 × 10⁻⁹ M 6.51 × 10⁻⁹ M h-EGF 0.05 μg/ml 0.005 μg/ml 0.05 μg/ml choleratoxin 1 × 10⁻⁹ M 1 × 10⁻⁹ M BSA-FAF 0.02mg/ml 0.02 mg/ml BPE 7.5 μg/ml 7.5 μg/ml Epinephrine 0.5 μg/ml 0.5 μg/mlRetinoic Acid 0.1 ng/ml 0.1 ng/ml GA-1000 Gentamycin Gentamycin 50 μg/ml5 μg/ml Amphotericin B Amphotericin B 0.05 μg/ml 0.05 μg/ml CaCL₂ BSAAscolbic Acid FCS FBS 5%

The invention also provides a normal regenerated tissue panel or chipconstructed by a characteristic 3-dimensional culture as well as aregenerated organ model body.

On a basal plate of a hard glass or quartz, a regenerated tissuedescribed above is formed, and its culture medium is allowed to flow.Multiple types of normal regenerated tissues can be in communicationwith each other as a channel for the culture medium. In the case of apanel or chip, the surface of the basal plate may be coated with asubstrate such as laminin or fibronectin.

We actually verified that it is possible to construct a normalregenerated tissue derived from the organs listed in Table 2 as well asa normal regenerated tissue derived from nerve, skin, mammary gland,liver, kidney and the like.

TABLE 2 Trachea and bronchia 1) Mucosa Epithelium: Non-ciliated cell,Ciliated cell, Goblet cell, Basal cell, Neurosecretory cell,Intermediate cell Tunica propria: Collagen fiber, Elastica, Fibroblast,Blood capillary Submucosa 2) Tracheal gland (Bronchial gland) Myxocyte,Serous cell 3) Terminal bronchiole Ciliated cell, Clara cell Alveoli 1)Alveolar septum Supporting tissue, Elastica, Collagen fiber, Cancellousfiber, smooth muscle Cells in alveoli: Interstitial cell (septal cell),fobrous cell, Contractile interstitial cell, phagocyte, mast cell,blood-derived cell 2) Alveolar pore 3) Blood capillary, Blood capillarypore, Endothelium, Pericyte 4) Alveolar epithelium Type I alveolarepithelium (alveolar squamous cell) Type II alveolar epithelium (largealveolar epithelium) Alveolar brush cell

Each panel or chip can be used as an organ model, which can be anestablished means for detecting the major effects and side effects of achemical such as a drug as well as an extract such as a hormone. Untilnow, commercially available bronchus, kidney, mammary gland, prostate,liver, nerve and skin or patient-derived cervical mucosa obtained ascells with an informed consent have been subjected to a 3-dimensionalculture and the regeneration of all tissues derived from various organshas been accomplished successfully. When all organs including heart andliver targeted by a drug and the like will be investigated, anapplication to a panel for assessing the major and side effects of thedrug will be enabled. It is a matter of course that an application to ameans for testing the sensitivity to an anti-cancer agent may bepossible.

By mixing the medium compositions for various panels and chips, acomfortable chemical assessment system will be established which doesnot only handle endocrinal hormones or autocrine or paracrine growthfactors but also handles nerves in near future.

For example in the case where the sensitivity of an anticancer agentalone or in combination with various radiotherapy by variousfractionations, a regenerated epithelium (7) in a stratified structureshown also in FIG. 1 is irradiated with a 20Gy or less or X-ray or γ-ray(9) for the purpose of suppressing its proliferation to form a novel FL,which is inoculated with cancer cells (at a density of about 1000cells/cm²) (10) which was separated from a surgical specimen, cut into1-3 mm square pieces and then maintained in a collagen gel prepared in aconditioning medium for the FL described above or a normal fibroustissue or with any other tissue containing a tumor interstitialmaterial. Then the medium is replaced with a medium whose growth factorlevels are as low as possible, and subjected to various treatmentsdescribed above after about 12 to 24 hours, followed by an MTT assayafter about 48 to 96 hours or gene expression profile, protein profileand the like after about 6 to 48 hours. When cancer cells (10) areadded, then the collagen gel is supplemented as desired, and theaddition of a chemical or an irradiation (11) may be employed to assessthe sensitivity of the cancer cells. Then at the time point when thecancer tissue is grown 3-dimensionally to a 3-5 mm-cube size or more,the collagen gel is further mounted to establish an environment close toa low oxygen condition, and then vascular endothelial cells aretransplanted on the outside of the collagen gel whereby enabling anapplication to the investigation of the angiogenetic ability ofindividual cancer species for the purpose of assessing an angiogenesisinhibitor. A known method for establishing a low oxygen condition in aculture medium may for example be a method which gives a spheroid cancercell population which is proliferated in a spherical shape like a greenalga ball by a rotational culture. In such a case, when the diameterbecomes about 200 to 400 micron or more, a low oxygen condition isestablished in the center. Based on these findings, it is possible alsoin the invention that a gel-encapsulated cancer tissue is proliferated3-dimensionally to establish a low oxygen condition in its center. Alsoby mounting a gel formed from a type IV collagen and the like and theninverting the entire structure, an application to a system forinvestigating invasion ability for the purpose of assessing a motilityor invasion inhibitor is possible.

Besides the assessment of the cancer cell sensitivity, a collagen gel(12) is supplemented on a epithelial cell layer in a stratifiedstructure for example as shown in FIG. 2, and then covered with a cancertissue (13), whereby enabling the assessment of the sensitivity of thecancer tissue to a chemical or irradiation.

On the other hand, a normal regenerated tissue having an epithelial cell(7) layer in a stratified structure shown in Figs. is supplemented ifnecessary with a collagen gel (12), whereby enabling the assessment ofthe sensitivity of the normal tissue to a chemical or irradiation.

With regard to the prediction of the major and side effects of apharmaceutical according to the invention, the use of a healthyhuman-derived normal regenerated tissue rather together with a normalregenerated tissue derived from a human having any pathologicalcondition such as a life style-related disease (in a sense of a humanhaving no cancer) may be of significance. In fact, it is preferable as aassessment system for the major and side effects on the cases ofdiabetes and the like.

For example, an inventive method described above can improve the truepositive rate by overcoming a clinically disadvantageous aspectassociated with a conventional collagen gel-employing histoculture drugresponse assay (HDRA) or CD-DST whose true positive rate is as low asabout 60% or less. On the contrary to the HDRA or CD-DST which is notregarded to be a recognized custom-made therapy which allows a mosteffective drug to be selected and which is disappointed by most ofclinical practitioners because of the fact that it can select aneffective drug at a probability as low as about 50%, an inventive methodlimits the concentration of an exogenous growth factor in a culturemedium to a low level and employs a normal tissue from an individualcase, whereby enabling a simultaneous detection of the side effects onthe normal tissue and the sensitivity of a cancer tissue coexisting withthe normal tissue, thus realizing a so-called custom-made cancertherapy. The inventive method can be applied also to a method forproducing a regenerated tissue or organ from a stem cell other than a TSwithout adding an excessive amount of a differentiation-inducing agentor an exogenous biological factor.

An inventive method is useful also as an in vitro assessment system fora gene therapy. While a two-dimensional culture system is employed forassessing the efficiency of a gene transduction and the like, it has agreat limitation. An inventive regenerated tissue as an actual organmodel is significant in terms of its 3-dimensional aspect.

Since a 3-dimensional culture of a normal tissue involves asignificantly rapid proliferation of fibroblasts which are abundant in asubmucosa and thus should be removed at a tissue level, it is coculturedto involve the following procedures.

1. A tissue mucosa taken upon surgery or biopsy is washed thoroughlywith a phosphate buffered saline (Ca ion- and Mg ion-free) containing anantibiotic (containing 100 units/ml penicillin, 0.1 mg/ml, 0.25 μg/mlFungizone).2. The sample is treated for about 24 hours at 4.0° C. with a DME(containing FCS) containing a dispase (100 units/ml).3. The epithelial layer and the submucosal layer are separated from eachother mechanically.4. Only the epithelial layer is treated for 30 minutes in a trypsinsolution (containing 0.025% and 0.05% EDTA (only when trypsin is poorlyeffective).5. The epithelial cells are separated by stirring with a magneticstirrer.6. The epithelial cells are collected by filtration through a nylon meshfilter.7. Similarly, the vascular endothelial cells and the fibroblasts areseparated from the submucosal layer for example by using a collagenasesolution.8. The cells thus obtained were sorted using monoclonal antibody labelsto isolate respective fractions.9. The supplemented factors such as a growth factor including serum arediluted to 1/10, or the culture is continued in a serum-free medium for3 days, whereby suppressing the proliferation of the fibroblast.

In a tissue level investigation readily applicable to a cancer tissue3-dimensional culture, a 1-3 mm-cube of a cancer tissue containing atumor interstice is produced from an excised cancer tissue, mounted on acollagen gel in a multi-well culture dish, and allowed to standpreferably until the normal tissue is grown 3-dimensionally to 4 layersor more.

With regard to human organ-derived cells (tissue), those shown in Tables3 and 4 are commercially available.

TABLE 3 Cell product Basal medium Supplemented factor Manufacturernumber product number product number Bronchial epithelium Santo JunyatuCo., Ltd CC-2540 CC-3119 CC-4124 (Clontics) Bronchiolar epithelium Sameas above CC-2547 CC-3119 CC-4124 Prostatic epithelium Same as aboveCC-2555 CC-3165 CC-4177 Mammary gland epithelium Same as above CC-2551CC-3151 CC-4136 Pulmonary microvascular Same as above CC-2527 CC-3156CC-4147 endothelium Human hepatocyte ASAHI TECNOGLASS CC-2591 CC-3198CORPORATION (Clontics) Human neutrophile Same as above CC-2599 CC-3209CC-4123 Cell Human proximal Same as above CC-2553 CC-3190 tubularepithelium Human skin-derived From Dr. ISHIZAKI SuSa Dulbecco 10% FBSfibroblast MEM Human lung-derived ASAHI TECHNOGLASS Normal/mg fibroblastCORPORATION fibroblast (Clontics) CC-2512 CC-3131 CC-4126 Humanumbilical Same as above HUVEC CC-3156 CC-4176 venous endothelium CC-2517—

TABLE 4 Cell name Cell product number Bronchial epithelium (NHBE)CCS-2540 Pulmonary microvascular endothelium CCS-2527 Pulmonaryfibroblast (NHLF) CCS-2512 Small airvay (SAEC) CCS-2547 Prostaticepithelium (PrEC) CCS-2555 Mammary gland epithelium (HMEC) CCS-2551Proximal tubular epithelial cell (RPTEC) CCS-2553 Mesangium cell (NHMC)CCS-2559 Neural progenitor cell (NHMP) CCM-2599 Human hepatocyte (Nheps)CCS-2591

Any of these cells is co-cultured to be used as a sample in theinvention. It is a matter of course that those listed above are notlimiting.

Attached FIGS. 3, 4, 5 and 6 are the photographs obtained in theinvestigation of a 3-dimensional culture of a commercial human-derivednormal tissue. These photographs correspond to those described below.

FIG. 3: The condition of a co-culture of normal pulmonary fibroblastsand vascular endothelial cells before the X-ray irradiation at 20Gy.

FIG. 4: The condition 24 hours after the transplantation of trachealepithelial cells 24 hours after the irradiation described above.

FIG. 5: The condition after 1 week.

FIG. 6: The condition of the stratified structure having 4 layers ormore although observed only partly.

Based on the examples described above, the formation of a normalregenerated tissue of the invention was evident.

FIGS. 7, 8 and 9 are those described below.

FIG. 7: The regenerated bronchial epithelial cells in a normalregenerated tissue by an inventive method.

FIG. 8: The regenerated bronchial epithelial cells 3 days after theX-ray irradiation at 10Gy.

FIG. 9: The regenerated bronchial epithelial cells 3 days after theX-ray irradiation at 20Gy.

As evident from these photographs, it is possible to assess thesensitivity of a normal cell tissue to an X-ray.

FIGS. 10, 11, 12 and 13 are those described below.

FIG. 10: The condition of regenerated bronchial epithelial cell layerformed by an inventive method which had been supplemented with acollagen gel and then transplanted with human pulmonary cancer-derivedAOI cells after three days.

FIG. 11: The condition immediately after the X-ray irradiation at 20GY 3hours after the transplantation similar to that in FIG. 10.

FIG. 12: The condition 3 days after the irradiation in the case shown inFIG. 11.

FIG. 13: The condition after the X-ray irradiation at 10Gy in the caseshown in FIG. 12.

As evident from FIGS. 10 to 12, it is possible to assess the sensitivityof pulmonary cancer cells to an X-ray and the anti-cancer effect of theX-ray irradiation. A carcinostatic agent may be assessed similarly.

Embodiment 2

Similarly to those described above, various human organ-derivedregenerated tissues were formed and subjected to the cancer therapysensitivity test.

1. Preparation of Mucosal Sheet 1.1 Preparation of SuSa (Feeder Layer)

As a feeder layer, SuSa (human fibroblasts) was employed. The culturemedium employed was a 10% foetal calf serum (Sigma chemical Co.,hereinafter referred to as FCS)-supplemented Dulbecco's Modified EagleMedium (Sigma chemical Co., hereinafter referred to as DEME). A 24-wellplate was inoculated at 5×10⁴ cells/cm² per well, and irradiated with20Gy of an X-ray after 24 hours.

1.2 Preparation of Epithelial Cells

The epithelial cells employed were renal proximal tubular epithelialcells (RPTEC), mesangium cells (NHMC), prostatic epithelial cells(PrEc), neural progenitor cells (NHMP), human hepatocytes (NHeps) andmammary gland epithelial cells (HMEC) (all from Clontech). The culturemedium formulations employed were a renal epithelial cell basal medium(Clontech, REBM) containing 6.51 ng/ml *TRIIODESILONINE*, 0.5 μg/mlepinephrin, 1 μg/ml GA-1000, 10 μg/ml transferrin, 5 μg/ml insulin, 0.5μg/ml hydrocortisone, a renal epithelial cell basal medium (Clontech,REBM), 0.01 μg/ml hEGF, 0.5% of FBS for the RPTEC, a mesangial cellbasal medium (Clontech, MsBM) containing 1 μg/ml GA-1000, 5% FBS for theNHMC, a prostate epithelial cell basal medium (Clontech, RrEBM)containing 13 μg/ml BPE, 5 μg/ml insulin, 1 μg/ml GA-1000, 0.1 ng/mlretinic acid, 10 μg/ml transferrin, 6.51 ng/ml *TRIIODESILONINE*, 0.5μg/ml hydrocortisone, 0.5 μg/ml epinephrin and 0.01 μg/ml hEGF for thePrEC, a neural progenitor basal medium (Clontech, NPBM) containing 0.01μg/ml hEGF-8, 0.01 μg/ml hEGF, 0.8% of NSF and 1 μg/ml 1GA-1000 for theNHMP, a hepatocyte basal medium (Clontech, HBM) containing 0.5 μg/mlhydrocortisone, 0.01 μg/ml hEGF, BSA, ascorbic acid, 10 μg/mltransferrin, 5 μg/ml insulin and 1 μg/ml GA-1000 for the NHEPS, and amammary epithelial basal medium (Clontech, MEBM) containing 5 μg/mlinsulin, 1 μg/ml GA-1000, 0.01 μg/ml hEGF, 13 μg/ml BPE and 0.5 μg/mlhydrocortisone for the HMEC. A collagen dish (IWAKI) was used for theincubation under 5% CO₂ at 37° C., and each strain was inoculated upon80% to 90% confluent at 5×10³ cell/cm² per well on the feeder layerdescribed above which had previously been provided using 6 24-wellplates per strain.

1.3 Regenerated Sheet Incubation

A mucosal sheet incubation was conducted using a DMEM (−). The culturemedium was supplemented with a 1% Anti-B, 10% fetal bovine serum (JRHBIOSCIENCES, hereinafter referred to as FBS), 5 μg/ml insulin, 5 μg/mltransferrin, 2×10⁻⁵ M *TRIIODESILONINE*, 1×10⁻⁹ M cholera toxin, 0.5μg/ml hydrocortisone, 10 ng/ml human epithelium growth factor (EGF:Takara), 1000/ml penicillin G (MEIJI SEIKA), 1 mg/ml kanamycin (Sigma,St. Louis, Mo., USA), and 2.5 μg/ml Fungizone (Gibco, Grand Iskl, andNY, USA). The incubation was conducted at 37° C. in a 10% CO₂ incubatorwith replacing the culture medium every two days. After removing theculture medium after 10 days, a collagen gel which was a 8:1:1 mixtureof a gel matrix type A gel, 10×MEM (NaOH-free) and a 0.05N—NaOH (NITTAGELATINE) containing 2.2% NaOH and 200 mM HEPES was applied in a volumeof 0.5 ml per well in the 24-well plate. After the incubation for 20days, the plates are divided to obtain duplicate samples for an MTTassay, and the RPTEC and the NHMC were inoculated with ACHN-L4, the PrECwith DU145, the NHMP with A7, the Nhep with Alex and the HMEC withMDA-MB-453, all at 5×10⁴/cm². 4 Hours after the inoculation, a CDDP wasadded and an X-ray irradiation was performed at 0Gy (control) and 0Gyafter 24 hours. 72 Hours after the irradiation, the MTT assay samplingwas performed.

2. MTT Assay

After the treatment with the CDDP, the culture medium was removed after72 hours, and the collagen gel was cut into about 3 mm-cube pieces,combined with a 0.2% collagenase, dissolved with shaking at 37° C.,centrifuged at 1000 rpm for 5 minutes to separate the cells, which werethen resuspended in the culture medium, which were then returned to a24-well plate. 0.5 mg/ml MTT labeling reagent (Roche Diagnostics,hereinafter designated as Roche) was added, and the plate was incubatedin a 10% CO₂ incubator at 37° C. for 4 hours, and a solubilizationsolution (Roche) in a volume equal to that of the culture medium wasadded and incubated in a 10% CO₂ incubator at 37° C. for 24 hours. Fromeach well, 200 μl aliquots were transferred to a 96-well plate, whichwas examined for the absorbance at the wavelength of 600 nm using amultiplate reader to obtain a measured value as an average of 3 wells.

3. Types of Tissues Derived from Organs Capable of being Regenerated

Any of the cells employed here, namely, the renal proximal tubularepithelial cells (RPTEC: FIG. 14), mesangium cells (NHMC: FIG. 15),prostatic epithelial cells (PrEc: FIG. 16), neural progenitor cells(NHMP: FIG. 17), human hepatocytes (Nheps: FIG. 18) and mammary glandepithelial cells (HMEC: FIG. 19), became a stratified structure within10 days after initiation of the culture. Most of them became a4-stratified structure within 3 weeks. Also since the stratifiedstructure had already been observed after 5 days which was earlier thanthe time when the gel was mounted for example on the mesangium cells(NHMC), it was verified that the collagen gel was not essential for theregeneration.

4. Results of Cancer Therapy Sensitivity Test

The results of the sensitivity test by the MTT assay are shown in FIGS.20 to 23. The designation gel+cancer in the figures corresponds to aconventional HDRA method. An inventive sensitivity test is representedby an epithelium+gel+cancer, while an epithelium+gel represents thesensitivity of a normal tissue. Since the cancer cells were notseparated exclusively, the MTT assay of the epithelium+gel+cancer gavethe results as a total of the cancer tissue plus the normal tissue whichwas irradiated at 20Gy and became a feeder layer.

In the cancer cells, a nude mouse-transplanted tumor and a clinicallyradiochemotheapy-resistant glioblastoma multiform derived A7 cellsexhibited a more resistance in the MTT assay than in the HDRA method(FIG. 20). The hepatoma cells Alex in the MTT assay exhibited nosignificant difference from the HDRA method, but the combination withcisplatin exhibited the difference in the effect. While the prostaticcancer cells DU145 in the MTT assay exhibited an extremely highsensitivity to the irradiation in the HDRA method, but it has beenrecognized clinically to be less sensitive in an experiment of a nudemouse-transplanted tumor test, resulting in a rather closer sensitivityobserved in the inventive method. Finally, it was revealed clinicallythat also in the nude mouse-transplanted tumor the radio-resistant renalcancer ACHN-L4 exhibited a irradiation sensitivity which differedbetween the mesangium cells NHMC- and the renal proximal tubularepithelial cells RPTEC-derived supporting tissues (FIGS. 22 and 23).Thus, these results are very interesting because they suggested that thesensitivity of identical cancer cells differed between the differentsupporting tissues. The cisplatin sensitivity indicated that theinventive sensitivity test gave the results which were closer to thoseobserved in the nude mouse-transplanted tumor. When the sensitivities ofvarious cancer cells observed in the novel sensitivity test are comparedin the MTT assay, the results observed in the ACHN-L4 cells which arehighly sensitive to cisplatin showed an agreement.

Embodiment 3

The cancer therapy sensitivity test was conducted in comparison with aconventional two-dimensional culture MTT assay system.

The results are shown in FIGS. 24 to 34. In these figures, a column ofthe cancer name (e.g: AOI) corresponds to the sensitivity of the cancercells in the two-dimensional culture. The second column (e.g.:AOI/(NHBE+AOI) corresponds to the sensitivity observed after treatingthe cancer tissue which had been subjected to a 3-dimensional culture onregenerated normal tissue followed by conducting the MMT assay usingonly the cancer cells. The third column (e.g.: NHBE/(NHBE+AOI))corresponds to the sensitivity observed after culturing only normalcells followed by co-culturing with the cancer tissue followed byremoving the cancer tissue to test only the 3-dimensionally regeneratednormal tissue.

Also in these figures, the brackets the MTT assay correspond, from theleft, the normal epithelial cell name employed (this time a bronchialepithelium (NHBE) or prostatic epithelium (PrEC)), the culture medium (1/10-fold or 1-fold), the fibroblast name employed as a feeder layer(human skin-derived (SUSA) or lung-derived (NHLF)). Control correspondsto the results of the MTT assay in a non-treatment group, X-ray to theresults of the MTT assay 72 hours after a single irradiation at 10Gy,5FU to the MTT assay data after the treatment with 5FU for 72 hours at10 μg/ml, 5FU-X-ray to the MTT assay data after the treatment with 5FUfor 3 hours at 10 μg/ml followed by the treatment with 5FU for 72 hoursconcomitantly with a 10Gy irradiation. The drug treatments with all ofthree drugs employed here were conducted at 10 μg/ml for 72 hours.

Typically, the drug treatments were conducted while comparing threeanti-cancer agents (CDDP, MMC, 5FU).

<A> Results with Skin-Derived Fibroblasts (SUSA) as Feeder Layer

1. As a result, the 3-dimensional normal tissue coexisting with thecancer tissue was revealed to be rather more resistant to theirradiation, MMC alone or MMC+X-ray when compared with the normal tissuealone, possibly because for example of the level of the growth factorwas 1/10 in the 1/10-level medium. It was also revealed that, whencompared with the 2-dimensionally cultured AOI lung cancer cells, the3-dimensional AOI cancer tissue coexisting with the normal tissue wasmore resistant to the MMC alone or MMC+X-ray.

2. The 3-dimensionally growing normal tissue was rather more resistantto the mitomycin MMC alone or MMC+X-ray, possibly because of thepresence of an excessive amount of the growth factor in the 1-fold levelculture medium. The 3-dimensional AOI cancer tissue was rather moreresistant than the two-dimensionally cultured AOI cancer cells, possiblybecause of the presence of the signals such as a growth factor from thenormal bronchial epithelium which was co-cultured and subjected furtherto a 10Gy irradiation.

3. The tendency described above was observed also with the anti-canceragent cisplatin. Especially in the 3-dimensionally cultured AOI cancercells, the degree of acquiring the resistance was influenced morepotently by the level (of the growth factor) in the culture medium whencompared with the two-dimensionally cultured AOI cancer cells. The testswith the irradiation or 5FU treatment in the 1/10-fold culture mediumrather suggested that the tendency of the promotion of the growth of theregenerated normal bronchial epithelial tissue after removing the AOIcancer tissue which had been co-cultured. Also with the normal tissuealone, it was suggested that the frequency of the side effects was low.In the 1-fold culture medium, the proliferation was rather promoted byeither of the irradiation or the treatment with 5FU alone possiblybecause of the presence of the added growth factor, but such a promotingeffect disappeared when the both was given concomitantly. The sideeffects on the normal tissue was suggested to be exerted rather potentlywhen the added growth factor level was high.

4. In the regenerated prostatic tissue in the 1/10-fold culture medium,the regenerated normal prostatic tissue after the removal of theprostatic cancer-derived DU-145 cancer cells irradiated at 10Gyexhibited a rather promoted proliferation. This cancer cells wererevealed to be highly sensitive to the irradiation similarly to the nudemouse transplantation. It was rather more sensitive when compared withthe two-dimensionally cultured cancer cells. When compared with the AOIcells, these cancer cells were rather resistant to cisplatin, highlysensitive to 5FU, and has a sensitivity to mitomycin similar to that ofthe AOI cells.

<B> Use of Lung-Derived Fibroblasts as Feeder Layer

1.1 An extremely different behavior was observed when using lung-derivedfibroblasts in the 1/10-fold culture medium.

Thus, no tissue exhibited any sensitivity to 5FU. To mitomycin, therewas no substantial difference between the skin-derived and lung-derivedfibroblasts.

2.1. An extremely different behavior was observed when using alung-derived fibroblast in the 1-fold culture medium.

Thus, while the difference in the sensitivity of the AOI cancer tissueto cisplatin by the difference in the fibroblasts was small, thetoxicity of cisplatin on the normal tissue was reduced. The3-dimensional culture resulted in a reduced sensitivity of the AOIcancer cell tissue and the normal tissue to 5FU. The sensitivity of the3-dimensional AOI cancer cells was increased. These results are well inagreement with the results observed when the same cancer cells weretransplanted into the nude mouse.

<C> Conclusion

1. The AOI tumor transplanted actually to a nude mouse exhibits aresistance so potent that a retarded growth was observed only forseveral days even when treated with cisplatin at 10 mg/kg (correspondingto 10 μg/ml in a culture system) which is so toxic that the body weightof the nude mouse is reduced to about ⅔. The AOI tumor was rather moresensitive to mitomycin than to cisplatin.

2. Based on the finding described above, it was revealed that in thesensitivity comparison using the 3-dimensionally cultured normal tissuesubstantiated that the sensitivity may vary greatly depending on whetherthe fibroblasts used as a feeder layer was derived from the identicalorgan or not. Thus, the sensitivity can correctly be detected bypreparing a feeder layer using an identical organ-derived fibroblast.

Since identical organ-derived fibroblasts can readily be available whenusing a clinical material, there is no problem. While an added growthfactor may influence the sensitivity markedly, there is still no problemsince the sensitivity was assessed rather correctly even when using a1/10-fold culture medium.

Based on the results of the experiment described above, the use of anectopic feeder layer (when using fibroblasts derived from the skin as afeeder layer for the lung cancer) may result in a marked variation inthe reaction (sensitivity). It is also considered that also since bothof the lung-derived fibroblasts (connective tissue) and the regeneratedbronchial epithelial tissue was employed eventually as feeders for thecancer, the natural sensitivity (reaction) was exhibited by the lungcancer-derived cells. Such findings are also supportive of the effect ofthe orthotopicity.

Embodiment 4

In EMBODIMENT 2, it was revealed that the collagen gel was not essentialfor the regeneration in inventive organ-derived tissue regeneration, andthese findings were verified also by using fibronectin which is one ofthe extracellular matrixes other than the collagen.

FIG. 35 shows the results for the kidney-derived cells: PrEc in thepresence only of the collagen, while FIG. 36 shows the results obtainedwithout using the collagen but using only 100 μg/ml of fibronectin. Asevident from these results, the formation of a stratified structure canbe accomplished also by fibronectin.

Embodiment 5

The organ-derived tissue regeneration by means of the stratifiedstructure of the invention was validated with regard to the effect ofthe addition of an extracellular matrix other than the collagen gel.

Thus, similarly to EMBODIMENT 1, human skin-derived fibroblasts as afeeder layer was covered with NHDF-Ad: human skin keratinized cells toform a stratified structure with adding fibronectin and laminin to thecollagen gel.

FIGS. 37( a), 37(b), 38(a) and 38(b) show the results.

As evident from these result, the addition of 2 μg/ml of fibronectin and1 μg/ml of laminin (fibronectin:laminin=2:1) (FIG. 28) to the collagengel gave the highest regeneration efficiency, and the ratios of 1:1 and1:2 were the next highest in this order (FIGS. 37( a) and 37(b)), andthe regeneration efficiency of the collagen gel alone (FIGS. 38( a) and38(b)) was revealed to be poorer than the formers.

Embodiment 6

5×10⁴ cells of a human melanoma-derived SK-Me126 cell were inoculated toa Boyden Chamber having a bed of 0.1 mg/ml MATRIGEL, and culturedtogether with the human keratinized epithelial cells NHDF-Ad in astratified structure on human skin-derived fibroblasts formed by aninventive method. By observing the cell invading the MATRIGEL to emergeout on the back, the invasion test was conducted. In this procedure, theratio of fibronectin and laminin added (F:L) to the collagen gel wasvaried similarly to EMBODIMENT 5. The results are shown in FIGS. 39( a)and 39(b). The invasion ability became highest also here when adding thecombination of 2 ng/ml or 1 ng/ml of the former with 1 ng/ml of thelatter.

Embodiment 7

The human prostate epithelium-derived PrEc cells on the 3rd day inEMBODIMENT 4 described above when altering the culture condition wereshown as photographs in FIGS. 40 to 42.

The even numbers correspond to the addition of 2.5 μl of fibronectin and2.5 μl of laminin to 500 μl of the collagen (F:L=1:1).

The odd numbers correspond to the addition of 5 μl of fibronectin and2.5 μl of laminin to 500 μl of the collagen (F:L=2:1).

Numbers 1 and 2 employed 250 μl/well of the type 1 collagen alone.

Numbers 3 and 4 employed 200 μl/well of the type 1 collagen togetherwith 50 μl/well of the type 2 collagen.

Numbers 5 and 6 employed 200 μl/well of the type 1 collagen togetherwith 50 μl/well of the type 3 collagen.

Numbers 7 and 8 employed 200 μl/well of the type 1 collagen togetherwith 50 μl/well of the type 4 collagen.

Numbers 9 and 10 employed 200 μl/well of the type I collagen togetherwith 50 μl/well of the type 5 collagen.

Numbers 11 and 12 employed 150 μl/well of the type 1 collagen togetherwith 25 μl/well of the type 2, 25 μl/well of the type 3, 25 μl/well ofthe type 4 and 25 μl/well of the type 5, thus employing 5 types intotal.

It was observed that any of the samples having odd numbers generallyexhibited an advanced stage of the statification, and the difference inthe collagen type was not reflected in the prostatic epithelium.

Embodiment 8

A human kidney cancer-derived ACHN-L4 was employed as cancer cells toconduct a cancer therapy sensitivity test. The results are shown inFIGS. 43 and 44. For an ACHN-L4/pilelayer, a Boyden chamber having a bedof 0.1 mg/ml of MATRIGEL on the stratified structure of each of humanrenal proximal tubular epithelial cells (RPTEC) and mesangium cells(NHMC) (Day 9) was employed. In the chamber, 5×10⁴ cells of the ACHN-L4were inoculated, and subjected to the irradiation after 24 hours or thedrug treatment for 72 hours followed by a therapy effect evaluation suchas an MTT assay. For a monolayer, a Boyden chamber having a bed of 0.1mg/ml of MATRIGEL on the RPTEC cells or the NHMC cells were employed. Inthe chamber, 5×10⁴ cells of the ACHN-L4 were inoculated, and subjectedto the irradiation after 24 hours or the drug treatment for 72 hoursfollowed by a therapy effect evaluation such as an MTT assay. A cancer(ACHN-L4) was subjected to the therapy sensitivity test similarly exceptfor using no underlying feeder layer, RPTEC or NHMC.

The X-ray dose employed here was 10Gy, and both of the CDDP at 10 μg/mland the MMC at 100 μg/ml were applied to the top and the bottom of theBoyden chamber.

The % viability was calculated from the difference in the absorbancebetween those at the wavelengths of 550 nm and 690 nm (X550-Y690).

FIG. 45 shows the change in the sensitivity of the human renal proximaltubular epithelial cells RPTEC in a mixed culture medium prepared byadding the culture supernatant of a skin keratinized cell-derivedNHDF-Ad cells (derived from heterotopic organ) in a stratified structure(After two days of culture incubation) to a fresh maintenance medium inthe ratios of 5:0, 4:1, 3:2 and the like.

FIGS. 46( a)-46(c) are photographs showing the condition in the casedescribed above.

In the experiments described above, it was verified that the MTT assayemploying a Boyden chamber enables an easy separation between the cancercells and the normal cells, whereby improving the quantitativity.

Also in view of the results of the MTT assay, the two-dimensionalculture (monolayer) and the 3-dimensional culture (pile-layer) exhibitedthe sensitivity to a therapy in the presence of a normal tissue whichwas different substantially even when comparing with conventional cancercells employed alone. It was also proven that the toxic profile observedin the normal tissue was different between the two-dimensional culture(monolayer) and the 3-dimensional culture (pilelayer). Also in the caseof the coexistence, the cancer cell reaction varies depending on thetype of the normal tissue derived from an identical human kidney.

It was also observed quantitatively that the cancer cell sensitivity wasaltered when the culture supernatant of a tissue in a stratifiedstructure derived from a different organ was mixed. Thus it was proventhat the circulation between the organs is of a great significance forexample in a toxicity test.

INDUSTRIAL APPLICABILITY

As detailed above, the invention overcomes the limitations and theproblems associated with the prior art described above, and make itpossible to obtain a regenerated tissue simply and reliably by a3-dimensional culture from a human-derived normal tissue as a base, andto provide, while utilizing the formers, a method for constructing asystem for assessing the effects and the side effects of a chemicalsubstance such as a pharmaceutical using a thus regenerated tissue as arespective organ model or a system for assessing the sensitivity and thelike.

1-30. (canceled)
 31. A normal regenerated tissue comprising a stratifiedstructure of epithelial cells on an irradiated feeder layer from anorgan derived fibroblast and vascular endothelial cells, wherein thefibroblasts, the endothelial cells and the epithelial cells arcorthotopic with regard to the originating organ, and wherein the organis not skin.
 32. The normal regenerated tissue according to claim 31,wherein the epithelial cells derive from nerve, oral mucosa, bronchus,mammary gland, liver, prostate or kidney.
 33. The normal regeneratedtissue according to claim 31, wherein the feeder layer consisting of alower layer of the fibroblasts and upper layer of the endothelial cells.34. The normal regenerated tissue according to claim 31, wherein theepithelial cells are in a stratified structure consisting of four ormore layers.
 35. The normal regenerated tissue according to claim 31,wherein the normal regenerated tissue is placed on a basal plate. 36.The normal regenerated tissue according to claim 31, wherein a culturemedium or culture fluid is allowed to flow in contact with a normalregenerated tissue.
 37. The normal regenerated tissue wherein a pluralof normal regenerated tissue according to claim 31 are allowed to passthrough the channel of the culture medium or culture fluid.
 38. Thenormal regenerated tissue which constitutes a regenerated organ modelbody of a normal regenerated tissue according to claim
 31. 39. Thenormal regenerated tissue according to claim 31, wherein the number ofvascular endothelial cells is one tenth of the number of fibroblasts.